Diabetic retinopathy remains the leading cause of blindness among young adults. We have shown that in the pathogenesis of diabetic retinopathy, matrixmetalloproteinase-9 (MMP-9) is activated in the retina via a small molecular weight G-protein, H-Ras, activated MMP-9 damages mitochondria and accelerates retinal capillary cell apoptosis, and MMP-9 gene knockout diabetic mice are protected from the development of retinopathy. Diabetes alters histone modifications, and MMP-9 is shown to be regulated by histone modifications in its promoter region. Our preliminary data show that in diabetes, the recruitment of p65 subunit of NF-kB is increased at retinal MMP-9 promoter and that of dimethylated lysine 9 of histone 3 (H3K9me2) is decreased, lysine-specific demethylase (LSD1) is activated and histone deacetylase (Sirt1) is inhibited, and overexpression of MnSOD protects diabetes-induced increase in MMP-9, histone modifications and alterations in the histone modifying enzymes. Furthermore, re-institution of normal glycemic control after a period of poor glycemic control in rodents fails to provide any benefit to MMP-9 activation, histone methylation and Sirt1 inhibition. Thus, our overall hypothesis is that in diabetes, retinal MMP-9 is regulated by histone modifications, and the enzyme remains elevated after termination of hyperglycemic insult and the retinopathy continues to progress. We propose to test this hypothesis methodically by addressing complementary questions proposed under three specific aims. The first aim will investigate the molecular mechanism by which diabetes activates retinal MMP-9, and will test the hypothesis that due to histone modifications, the recruitment of p65 at MMP-9 promoter is increased resulting in its activation'. Since oxidative stress is considered to play an important role in the development of diabetic retinopathy, in the second aim, we will investigate the role of oxidative stress in the regulation of MMP-9. Our working hypothesis predicts that due to increased oxidative stress, Sirt1 is inhibited and deacetylation is decreased, and this facilitates the binding of p65 at the MMP-9 promoter. The third aim will determine the role of MMP-9 in the metabolic memory phenomenon associated with the progression of diabetic retinopathy, and will test the hypothesis that due to continued histone modifications, MMP-9 remains active after reversal of hyperglycemia, and the dysfunctional mitochondria continues to accelerate apoptosis of capillary cells. These proposed studies are based on compelling preliminary data generated using valid in vitro and in vivo model systems. The central hypothesis will be tested using active plasmids, siRNAs and pharmacological inhibitors in isolated cells, and in vitro findings will be validated in in vivo models using genetically manipulated mice and in retinal microvessels from human donors with diabetic retinopathy. The results are expected to demonstrate the role of histone modifications in retinal MMP-9 activation in diabetes, and in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. This should reveal novel targets for therapies, including regulators of histone modifying enzymes, to prevent retinopathy in the early stages of its development, and in its progression after termination of hyperglycemia. This will offer patients additional therapeutic means to prevent/retard the sight-threatening complication of diabetes.